Cargo Sorting and Intralumenal Vesicle Budding by the ESCRT Complexes Membrane budding and fission is a fundamental process of eukaryotic cell biology. Endocytosis, the formation of intracellular transport and secretory vesicles, and mitochondrial fission are examples of inward budding. In the classical example of clathrin-mediated endocytosis, the cytosolic protein dynamin forms arrays on the outside of the membrane neck, and membrane fission is driven thermodynamically by the hydrolysis of GTP. The formation of multivesicular bodies (MVBs) is the prototypical example of outward budding. MVBs are formed during the maturation of endosomes destined to fuse with lysosomes, and mediate the sorting of ubiquitinated membrane proteins to the lysosome. Portions of the limiting membrane of the endosome are internalized to form intralumenal vesicles (ILVs). When the MVB fuses with the lysosome, ILV contents are degraded by lysosomal hydrolases. When ILVs are released through fusion with the plasma membrane, they are referred to as exosomes. The budding of enveloped viruses from the plasma membrane and cell division (cytokinesis) are other examples of outward budding events. Outward budding events in MVB formation, viral budding, and cytokinesis are directed from the cytosol. Since cytosol is in contact with the inside, not the outside of the neck of the nascent bud, the mechanics of membrane fission differ fundamentally from inward budding, and utilize a completely distinct protein machinery. A major breakthrough in understanding outward budding came from the identification in yeast of the ESCRT machinery responsible for MVB formation. The ESCRT machinery is conserved throughout eukaryotes, and many enveloped viruses of mammals use the ESCRT pathway to bud, including HIV-1. The closure of the membrane neck in cytokinesis also uses the ESCRT pathway. The assembly of ESCRT complexes on endosomes is triggered by the presence of phosphatidylinositol 3-phosphate (PI(3)P) and ubiquitinated cargo proteins. ESCRT-I and II directly bind to cargo, and in turn recruit ESCRT-III. There are four ESCRT-III subunits in yeast, Vps2, Vps20, Vps24, and Snf7, together with two associated ESCRT-III-like proteins, Did2 and Vps60. ESCRT-III subunits exist in the cytosol as monomers, and assemble with each other on membranes in large multimeric arrays. ESCRT-II is a Y-shaped complex that contains two copies of the Vps25 subunit, which recruits ESCRT-III by directly binding to Vps20. Vps20 binds to Snf7, comprising a subcomplex of ESCRT-III. Snf7, in turn, directly binds to the Bro1 domain of the ESCRT-associated protein Alix (known as Bro1 in yeast). The Vps20:Snf7 complex recruits the Vps2:Vps24 subcomplex to form the complete ESCRT-III complex. A subset of ESCRT-III proteins directly bind to the N-terminal MIT domain of the AAA ATPase Vps4. Vps4 is a central player in the MVB pathway that is required for the disassembly of the ESCRT-III complex. ESCRT function can be conceptually separated into two phases: cargo recruitment and concentration, followed by membrane invagination and budding. The long term objectives of this project are to: 1) determine the structures of ESCRT complexes by x-ray crystallography, abetted where necessary by electron microscopy, hydrodynamics, molecular simulations, and small angle x-ray scattering; 2) to determine how ESCRTs assemble on membranes containing PI(3)P and cargo using binding and spectroscopic techniques; and 3) to study the mechanism of ILV formation by a microscopic, spectroscopic, and structure/function approaches. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the UEV and Vps28 C-terminal (CTD) domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I.In FY2011, we determined a low resolution structure of ESCRT-I in solution. In FY2012 we built on this study by determining the solution structure of supercomplex formed by ESCRT-I and -II together. This is the key assembly responsible for stabilizing the neck of the membrane bud. The structural ensemble was cross-validated against single molecule Frster resonance energy transfer (FRET) spectroscopy, which suggested the presence of a continuum of open states. This study led to the most detailed model for ESCRT-mediated budding and scission to date. The physical mechanism of membrane budding by ESCRTs has been unresolved and a subject of controversy. Spurred by theoretical modeling, we tested whether ESCRTs could induce lateral lipid phase separation. Theory predicts that line tension arising between different lipid domains could promote budding. Using a supported bilayer model, we found that human ESCRT-II could assemble on supported bilayers into small clusters that induced a liquid ordered lipid domain that was not pre-exising, even in lipid mixtures that are far from the phase boundary for spontaneous segregation. The clusters bind stoichiometrically to ubiquitin and to the ESCRT-III protein VPS20. This finding helps connect theoretical analysis of ESCRTs to their biology and so clarifies the budding mechanism. Human ESCRT-I functions at multiple loci in the cell, including both endosomes and the plasma membrane. Human ESCRT-I is not a single entity, but rather a heterotetramer containing one copy each of TSG101, VPS28, VPS37 isoform A-D, and either MVB12 A-B or UBAP1. We found that the MABP domain of MVB12A and B binds to acidic lipids found at both the endosome and plasma membrane, and solved its crystal structure. This helps explain how the MVB12-containing forms of ESCRT-I can function both in endosomal cargo sorting and in plasma membrane processes such as cytokinetic abscission, HIV-1 budding, and exosome formation and release.